A proton current drives action potentials in
genetically identified sour taste cells
Rui B. Chang, Hang Waters, and Emily R. Liman1
Department of Biological Sciences, Section of Neurobiology, University of Southern California, Los Angeles, CA 90089
Edited* by King-Wai Yau, The Johns Hopkins School of Medicine, Baltimore, MD, and approved October 29, 2010 (received for review September 16, 2010)
Five tastes have been identified, each of which is transduced by
a separate set of taste cells. Of these sour, which is associated with
acid stimuli, is the least understood. Genetic ablation experiments
have established that sour is detected by a subset of taste cells that
express the TRP channel PKD2L1 and its partner PKD1L3, however
the mechanisms by which this subset of cells detects acids remain
been hindered because sour responsive cells represent only a small
fraction of cells in a taste bud, and numerous ion channels with no
role in sour sensing are sensitive to acidic pH. To identify acid-
sensitive conductances unique to sour cells, we created genetically
under the control of the PKD2L1 promoter. To measure responses
to sour stimuli we developed a method in which suction electrode
recording is combined with UV photolysis of NPE-caged proton.
Using these methods, we report that responses to sour stimuli are
instead are mediated by a proton conductance specific to PKD2L1-
expressing taste cells. This conductance is sufficient to drive action
potential firing in response to acid stimuli, is enriched in the apical
membrane of PKD2L1-expressing taste cells and is not affected by
targeted deletion of the PKD1L3 gene. We conclude that, during
sour transduction, protons enter through an apical proton conduc-
tance to directly depolarize the taste cell membrane.
vides distinct information on the nutritional content and safety of
ingested food and is detected by a separate subset of cells within
the taste bud (1). Although great strides have been made in
understanding the sensory transduction cascade involved in bit-
ter, sweet, and umami taste, and more recently in salty taste (2),
relatively little is known about sour. Recent studies using genetic
ablation have clearly demonstrated that sour taste is detected
by a subset of taste cells defined by expression of the TRP ion
channel PKD2L1 (3). However, the ionic mechanisms that un-
derlie sensory responses to sour remain poorly understood.
Substances that taste sour are acidic and include fermented
foods and unripe fruits. Psychophysical studies in humans have
shown that sour is elicited by solutions containing HCl at con-
centrations >1 mM (pH 3) (4), suggesting that an acidic extra-
cellular pH is a stimulus for sour. In addition, it has been pro-
posed that intracellular acidification contributes to sour taste (5,
6), as weak acids that induce intracellular acidification, such as
acetic acid, produce a more intense sour sensation than do strong
acids (4). Consistent with a role for extracellular protons in sour
sensing, it was recently shown that sour responses elicited by CO2
are dependent not on intracellular conversion of CO2 to bi-
carbonate (which releases a proton), but rather on extracellular
conversion of CO2to bicarbonate (7). Several candidate mole-
cules have been proposed to mediate detection of sour, including
acid-sensing ion channels (ASICs), hyperpolarization activated
channels (HCNs), two-pore domain K+channels, and most re-
cently the PKD2L1/PKD1L3 heterodimer, a member of the tran-
sient receptor channel family (8–15). However, direct evidence
linking any of these receptors to sour taste is still lacking (16).
Notably, although an initial report showed that the PKD2L1/
ost vertebrate species are responsive to five basic tastes:
sweet, bitter, umami, sour, and salty, each of which pro-
PKD1L3 heteromer was activated by exposure to solutions of low
pH (pH <3) (14), subsequent reports show that activation occurs
at a delay upon removal of the acid (17, 18). Moreover, a recent
study of mice carrying a targeted deletion of PKD1L3 found
no significant deficits in behavioral or nerve responses to sour
The study of sour taste has been problematic for two reasons:
First, the taste bud is heterogeneous and only ∼20% are ex-
pected to be sour responsive (3, 13, 14); and second, many ion
channels with no role in sour sensing are either blocked or ac-
tivated by acidic pH (20). Thus, at present no study has docu-
mented the presence of a proton-sensitive conductance that is
specific to sour-responsive taste cells and is therefore a candidate
to mediate sour transduction. To identify such a conductance,
we generated mice in which yellow fluorescent protein (YFP) is
driven by the promoter of PKD2L1. To identify responses specific
to this subset of cells, we also measured responses from cells that
express GFP driven by the promoter of TRPM5, a downstream
element of the signal transduction cascade, and a marker for cells
responsive to bitter, sweet, and umami tastes (21–23). By com-
paring responses from YFP-labeled cells to responses from GFP-
labeled cells, we have identified a proton conductance that is
specific to sour-responsive taste cells. Using a unique method of
UV-uncaging of protons at the apical surface of the taste cell,
combined with suction electrode recording, we demonstrate that
proton entry through this conductance is sufficient to initiate the
Functional Responses to Sour of Genetically Labeled Sour Taste
Receptor Cells. PKD2L1 is expressed in a small subset of taste
receptor cells that are required for nerve responses to sour stimuli
(3). Using BAC modification (24) similar to that previously used
to target PKD2L1 cells for genetic ablation (3), we generated
mice in which the promoter of PKD2L1 drives expression of
YFP (Fig. S1A). To compare functional properties of PKD2L1-
expressing cells with those of TRPM5-expressing cells, we used
a line of mice in which the TRPM5 promoter drives the expres-
sion of GFP (22, 23).
To determine whether YFP faithfully identifies the population
of PKD2L1-expressing cells, we stained sections of tongue con-
taining circumvallate papillae (Fig. 1 A and B) with a polyclonal
antibody directed against mPKD2L1. We observed faithful coex-
pression of YFP with PKD2L1, and complete exclusion of YFP
from taste receptor cells that express TRPM5. YFP could also
be detected with epifluorescence in dissociated taste receptor
cells from circumvallate, follate, and fungiform papillae (Fig. 1C).
Single-cell RT-PCR of singly dissociated taste cells showed that
YFP-positive taste receptor cells expressed PKD2L1 as well as
Author contributions: R.B.C., H.W. and E.R.L. designed research; R.B.C, H.W., and E.R.L.
performed research; R.B.C., H.W., and E.R.L. analyzed data; and R.B.C. and E.R.L. wrote
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
See Commentary on page 21955.
1To whom correspondence should be addressed. E-mail: liman@USC.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| December 21, 2010
| vol. 107
| no. 51 www.pnas.org/cgi/doi/10.1073/pnas.1013664107
SNAP-25, as expected for this subset of cells (22, 25) but not
TRPM5 (Fig. S1B). Moreover, patch clamp recording confirmed
that PKD2L1-YFP cells have the functional properties expected
for this subset of cells, including expression of voltage-gated Ca2+
channels (25, 26) (Fig. S2). These results clearly demonstrate that
YFP expression can be used to identify PKD2L1-expressing taste
cells in this BAC transgenic mouse line.
Acid-evoked action potentials (APs) have previously been
detected in a subpopulation of taste cells that express GAD67
(27), which identifies the same population of cells as does
PKD2L1 (25). To determine whether YFP-positive cells from
PKD2L1-YFP mice respond to sour stimuli, we examined the
responses of dissociated cells to acids using loose patch record-
ings. A 25-mM quantity of KCl, which is expected to depolarize
the cell to ∼ −40 mV, was used to select for cells capable of firing
action potentials, and to exclude unresponsive cells from our
analysis. Remarkably, all YFP cells from the PKD2L1-YFP mice
that responded to KCl (13/30 cells) also responded to HCl (pH 6;
13/13 cells) and, where tested, responded to acetic acid (10 mM,
pH 6; 3/3 cells; Fig. 1 D and E). Each acid stimulus evoked a burst
of ∼6 APs within the first 2 s. In some cells, the responses to acids
could be repeated more than five times with little or no desen-
sitization. In contrast, KCl-responsive GFP-positive cells from
TRPM5-GFP mice did not respond to HCl or acetic acid (n = 6
and n = 4; Fig. 1E).
Together these results demonstrate that in our PKD2L1-YFP
mouse line, YFP faithfully identifies PKD2L1-expressing cells.
Moreover, the results show that PKD2L1-expressing cells are
specifically responsive to extracellular acidification.
An Inward Current Is Specifically Activated in PKD2L1-Expressing Cells
by Low Extracellular pH. To understand the ionic basis for the
generation of action potentials in response to acids by PKD2L1-
5 acetic acid (2 mM) and pH 5 HCl [buffered with 2-(N-morpho-
in PKD2L1-expressing cells, and the average magnitude of the
currents elicited by the two stimuli were no different (150.1 ± 24.2
pA versus 113.7 ± 18.0 pA at −80 mV; n = 7; P = 0.28; Fig. 2A).
Moreover the total current measured in the presence of either
stimulus, consisting of a linear inward component and a rectifying
outward component, reversed at a similar voltage (26.7 ± 2.1 mV
versus 28.2 ± 3.7 mV; n = 6–10; Fig. 2 B and C); because Mes
cannot produce intracellular acidification, these data argue that,
under our conditions, intracellular acidification does not con-
tribute to the activation of an inward current. To determine
whether a component of current was lost as a result of the enzy-
matic treatment of the cells, we measured responses under the
same conditions from non-enzymatically dissociated taste cells.
No differences in the magnitude or other characteristics of the
current were observed (Fig. 2C). In the previous experiments, we
used a Cs+-based intracellular solution to reduce contributions
from pH-sensitive K+currents (28, 29). We also measured
responses from PKD2L1-expressing cells to acids with K+in the
pipette. The magnitude of the inward current elicited by acetic
acid or HCl, pH 5 (81.1 ± 28.5 pA, n = 4 and 176.5 ± 46.9, n = 3,
respectively) was no different with K+in the pipette as compared
with Cs+(P = 0.11 and P = 0.19, respectively). In TRPM5-
expressing cells, pH 5 HCl evoked only a small inward current of
11.5 ± 6.0 pA at −80 mV (Fig. 2C), indicating that the acid-
evoked inward current is specific to PKD2L1-expressing cells.
PKD2L1 forms a complex with PKD1L3 when expressed in
HEK293 cells, and this complex has been proposed to form a
sour receptor (14). To determine whether the acid-induced cur-
rent we observed in sour cells is conducted by the PKD2L1/
PKD1L3 channel, we crossed our BAC transgenic mice with
PKD1L3 KO mice (19) and measured responses to acids from
YFP positive, PKD1L3−/−taste cells. Notably, neither the am-
expressed by taste cells that are immunoreactive for PKD2L1 (red, A), but not
by cells immunoreactive for TRPM5 (red, B). (C) Acutely dissociated taste cells
from PKD2L1-YFP mice showed intense YFP fluorescence. (D) Action potentials
recorded from a PKD2L1-YFP cell (Upper) and a TRPM5-GFP cell (Lower). Sol-
utions at pH 6 were buffered with either 10 mM Mes or 10 mM acetic acid
(HOAc). (E) Average data from experiments as in D. Control (con) was mea-
sured in the 10-s interval before KCl (25-mM) stimulation. Data represent
mean ± SEM; **P < 0.01; ***P < 0.001. (Scale bars: A and B, 10 μm; C, 5 μm.)
YFP expression driven by the PKD2L1 promoter faithfully marks sour-
PKD2L1-YFP cells. (A) Whole-cell current at −80 mV from a PKD2L1-YFP taste
cell in response to HCl pH 5 (10 mM Mes) or HOAc, pH 5 (2 mM) in the
presence of extracellular Na+. (B) Replacement of extracellular Na+with
NMDG+did not affect the inward current at −80 mV in response to pH 5
(HCl). I-V relationship measured at the time points indicated are shown at
Right. (C) Average magnitude of the acid-induced current at −80 mV from
experiments as in A and B. Average reversal potential (Er) of the total cur-
rent under each condition is shown at Right (n = 3–15). Note that the re-
sponse is indistinguishable between HCl (Mes) and HOAc, and manipulations
of the major ions (Na+, Ca2+, and Cl−) had no effect (P > 0.17 for all com-
parisons of current magnitude with control and P > 0.14 for all comparisons
of Er with control). Data represent the mean ± SEM. **P < 0.01.
Extracellular acidification specifically evokes an inward current in
Chang et al.PNAS
| December 21, 2010
| vol. 107
| no. 51
plitude nor the time course of the current evoked was signifi-
cantly different between PKD1L3−/−and WT cells (Fig. 2C).
Moreover, heterologously expressed PKD2L1/PKD1L3 did not
generate an acid-activated current under the same conditions
used for recording responses from taste cells (Fig. S3). Thus the
acid-induced current that we recorded from sour cells is not
mediated by the PKD1L3/PKD2L1 complex. Together these data
establish that an acid-evoked inward current, sensitive to extra-
cellular pH,is specifically expressed by sour-responsive taste cells.
Inward Current Activated by Acid in PKD2L1-Expressing Cells Is
Carried by Protons. Many types of ion channels have been pro-
posed to mediate sour taste transduction. To understand which,
if any, of these contribute to the inward current evoked by ex-
tracellular protons in sour cells, we examined the ionic selectivity
of the evoked current. Surprisingly, when we replaced Na+in
the external solution with either of the two large cations, N-
methyl-D-glutamine (NMDG+) or tetraethylammonium (TEA+),
both of which are impermeable through nearly all known cation
channels, neither the amplitude (measured at −80 mV) nor the
reversal potential of the acid-induced inward current was signif-
icantly altered (Fig. 2 B and C). Moreover, lowering the con-
centration of external Ca2+from 2 mM to 40 μM, where TEA+
was the only other external cation, had no effect on the amplitude
of the inward current or reversal potential. Thus the inward
current is not carried by either of the major cations, Na+or Ca2+.
An inward current at −80 mV could also be mediated by Cl−
efflux. To test this possibility, we increased the concentration of
Cl−in the pipette from 20 mM to 150 mM, which should increase
the efflux and hence the magnitude of the inward current if car-
ried by Cl−; again, the amplitude and reversal potential of the
inward current activated by acid was unchanged (Fig. 2C). As
this rules out possible contributions from external Na+and Ca2+
and from intracellular Cl−, the only remaining possible charge
carrier is H+.
To determine whether the acid-induced inward current was
carried by protons, we measured the effect of changing the ex-
ternal pH. Responses were first evident at pH 6, which corre-
sponds to a proton concentration of 1 μM, and the amplitude of
the response increased as the extracellular proton concentration
increased (Fig. 3 A and B). To examine whether the current can
be attributed to proton entry, we measured the reversal potential
of the current at varying pH. We observed a large shift in the
reversal potential (Er) of the current toward positive values as
the proton concentration increased (51 mV/pH; Fig. 3 A and C),
consistent with a contribution of a proton-permeable conduc-
tance to the acid-evoked current. It should be noted that we
couldnot subtract background
assumptions about their pH sensitivity. The observation that the
reversal potential of the total current was consistently less than
that predicted from the Nernst equation for a purely proton-
selective conductance is consistent with contributions from ei-
ther cationic or anionic conductances.
To further characterize the proton current in PKD2L1-
expressing cells, we examined the effect of a number of pharma-
cological agents known to block proton conductances. Proton
currents through amiloride-sensitive ion channels have been
proposed to play a role in sour taste transduction (30); however,
amiloride did not block the proton current in PKD2L1-YFP cells
(Fig. 3D). In addition, the following agents that affect various
mM Cd2+(an inhibitor of proton currents); 100 μM desipramine
(an inhibitor of a proton permeable serotonin transporter); and
200 nM Bafilomycin A1 (an inhibitor of the proton-permeable c
subunit of V-ATPase) (31) (Fig. 3D). Only zinc, which blocks
the recently identified proton channel Hv1 as well as many other
channels (31–33), produced a significant block of the current. At
1 mM, zinc produced near-complete block of the acid-evoked in-
the block was incomplete (20%, n = 2).
To confirm that proton entry occurs specifically in PKD2L1-
expressing cells, we performed experiments in which the intra-
cellular pH was monitored by a fluorescent pH indicator, car-
boxy-DFFDA (Fig. 3 E and F). As expected, extracellular pro-
tons were not able to cross the cell membrane in the absence of
a specific transport mechanism, as no intracellular acidification
was observed in response to HCl pH 5 in HEK293 cells or
TRPM5-GFP taste cells. In contrast, the same acid solution
(Fig. 3 E and F), consistent with the idea that a proton entry
pathway exists specifically in these cells. All cells were acidified
by acetic acid, which in its undissociated form can permeate the
Proton Entry Can Drive Action Potential Generation in PKD2L1-YFP
Taste Receptor Cells. The preceding data show that we have iden-
tified a proton current that is specifically expressed by PKD2L1-
expressing taste cells and that is therefore a candidate to mediate
sour transduction. However, because strong acids are not readily
permeable through the tight junctions of the lingual epithelium
(34), to participate in transduction this proton current must be
located on, although not necessarily restricted to, the apical sur-
face of the cell. We also expect that if this current contributes to
transduction, apical entry of protons should be sufficient to drive
protons. (A) Current–voltage relationship from a PKD2L1-YFP cell in re-
sponse to pH 6, 5.5, and 5 (HCl) solutions under Na+-free conditions. (B and
C) Average magnitude (at −80 mV) and reversal potential of the currents
from experiments as in A (n = 3–15). Dashed line shows a linear fit of the
data with a slope of 51.0 ± 3.8 mV/pH. (D) Effect of indicated chemicals on
current evoked by pH 5 (HCl) at −80 mV. Concentrations were as follows:
1 mM CdCl2; 100 μM desipramine; 200 nM Bafilomycin A1; 1 mM ZnCl2; and
30 μM amiloride. Block was measured as magnitude of current after in-
cubation with test chemical for 10 s, as compared with magnitude of current
immediately before application. No chemicals were given in the control
group, and the ∼10% block represents normal rundown of current in 10 s.
(E) Change in intracellular pH, as indicated by fluorescence intensity of
carboxy-DFFDA, in response to pH 5 (HCl) and pH 4 (20 mM HOAc) solutions
in a PKD2L1-YFP (Upper) and a TRPM5-GFP (Lower) taste cell. (F) Average
data from experiments as in E. Data represent mean ± SEM. ***P < 0.001.
The acid-evoked inward current in PKD2L1-YFP cells is conducted by
| www.pnas.org/cgi/doi/10.1073/pnas.1013664107Chang et al.
firing of action potentials and to produce an elevation of in-
tracellular Ca2+. To address these points, we developed a method
for rapidly elevating proton concentrations within a restricted
cellular domain, based on UV uncaging of an NPE-caged proton
(Fig. 4A). We further used the suction electrode recording
method, which has been useful in elucidating mechanism of visual
and olfactory transduction (35, 36). With this method, ionic cur-
rents can be measured from the portion of the cell that has been
drawn into the pipette, and the capacitive currents produced
by action potentials can be simultaneously detected, regardless
of their location. In control experiments, we found that when
loaded into the pipette, NPE-caged proton could, upon photoly-
sis, activate TRPV1 channels and ASIC channels in cell-attached
recording (Fig. 4B).
We first examined responses of taste cells when the apical
region of PKD2L1-YFP cells was drawn into the pipette (Fig.
4A) and protons were uncaged in the pipette. A large receptor
current was observed under these conditions, which was accom-
panied by a burst of action potentials (Fig. 4C; action potentials
were observed in seven of eight cells). The response was specific
toPKD2L1-expressing cells,asUVuncaging ofprotonsproduced
only a small receptor current and no action potentials in TRPM5-
expressing cells under identical conditions (Fig. 4C). The recep-
tor current and accompanying action potentials in PKD2L1-
expressing cells were completely blocked by 10 mM Hepes in the
pipette, confirming that they were elicited in response to the
changes in concentration of extracellular protons (Fig. 4C). As
expected if protons carry the transduction current, there was no
difference in the magnitude of the current or the rate of action
potentials when NMDG+replaced Na+in the pipette solution,
and the responses were completely abolished in the presence of
proton-channel blocker Zn2+(1 mM) (Fig. 4C).
To determine whether proton channels were enriched on the
apical surface of the taste cells, we measured responses from the
basolateral surface of PKD2L1-expressing cells. The acid-evoked
current recorded from the basolateral surface was significantly
smaller than the current recorded from the apical surface, and
fewer action potential were evoked in this configuration (Fig.
4C), consistent with an enrichment of the proton current on the
apical surface; however, we cannot rule out the possibility that
differences in responses may be partly attributable to differences
in the amount of membrane drawn into the suction electrode.
Finally, we tested a series of chemicals for their ability to block
the acid-evoked current and acid-evoked action potentials. No
significant effect was observed of the anion transporter blocker
blocker DCCD (200 μM), or the Cl−channel blocker NPPB (100
the latter two blockers (Fig. S4).
Ca2+imaging has been used extensively to characterize re-
sponses of taste cells to sensory stimuli, including sour substances
(6, 37). To determine whether the receptor current measured in
PKD2L1-expressing cells in response to uncaging of NPE-caged
protons is accompanied by an elevation of intracellular Ca2+, we
of intracellular Ca2+was observed in response to UV uncaging of
NPE-caged protons on the apical surface of the cells (Fig. 5 A and
two cells that generated action potentials, even though these cells
voltage-gated Ca2+channels (25, 26).
Our results provide evidence that the sensor for strong acids in
sour taste cells is an apical proton conductance that is specific to
PKD2L1-expressing taste cells. Previous experiments have also
found evidence for proton influx during sour transduction. For
example, receptor potentials in frog fungiform taste cells in re-
sponse to acetic acid were partially blocked by the proton pump
blocker DCCD (38), and responses in hamster taste buds to citric
acid under Na+-free conditions were attributed to proton influx
through amiloride-sensitive ENaCs (30). Other evidence for
proton influx into taste cells comes from studies showing changes
in intracellular pH in the intact taste bud in response to HCl,
although sour-responsive cells were not distinguished from other
types of taste cells in these studies (37, 39). In the present report,
we show that a Zn2+-sensitive H+conductance is selectively
expressed in genetically identified sour taste cells, and we dem-
onstrate the necessity of this current for sensing of strong acids
by these cells. The molecular identity of the channel or trans-
porter that underlies this current is presently unknown, as the
proton conductance that we identified does not show the recti-
fication properties expected of the recently identified voltage-
gated proton channel, Hv1 (32, 33).
A number of mechanisms have previously been proposed
to contribute to sour sensation, including activation of Na+-
permeable channels or block of K+channels by intracellular or
extracellular protons. A similarly diverse set of candidate re-
ceptor molecules have been identified that include acid-sensing
ion channels, hyperpolarization activated channels, two-pore
evokes a receptor current and action potentials. (A) Apical mem-
pipette containing 2 mM NPE-caged proton. (Scale bar, 10 μm.) In
this mode, the cell membrane is intact and acid stimuli can be se-
lectively delivered to the apical surface. (B) Control experiments
cell-attached mode evoked endogenous ASIC currents in untrans-
fected HEK 293 cells (Upper) and TRPV1 currents in HEK 293 cells
transfected with TRPV1 (Lower). (C) Membrane current and action
potentials recorded from taste cells upon UV uncaging of NPE-
caged proton in pipette as in A. PKD2L1-YFP, but not TRPM5-GFP,
cells responded to apical delivery of protons with a large inward
current and the generation of a train of action potentials. Other
traces show effects of changing the composition of the pipette
are shown at Right. Blue bars represent magnitude of currents
evoked by the UV flash; red bars represent the rate of evoked ac-
tion potentials. Data represent mean ± SEM. **P < 0.01, ***P <
0.001, as compared with responses from apical surface with Na+in
pipette except for Zn2+, for which the control was with NMDG+in
the pipette. Responses with NMDG+in the pipette were not sig-
0.64 and P = 0.13 for current and rate of APs, respectively).
Chang et al. PNAS
| December 21, 2010
| vol. 107
| no. 51
domain K+channels and transient receptor potential channels
(9, 10, 13–15, 28–30). We have found no evidence for a contri-
bution to sour taste of proton-gated Na+-permeable channels
such as ASIC or HCN channels, as extracellular protons did not
activate an Na+-permeable conductance in PKD2L1-expressing
cells, and apical delivery of protons elicited action potentials
in the absence of Na+ions in the apical solution. Moreover, the
ENAC channel blocker amiloride had no effect on the response
of sour taste cells to bath or apically applied acids. Weak acids,
such as acetic acid, have been proposed to elicit sour responses
by acidifying the cell cytosol, blocking potassium channels, and
thereby causing membrane depolarization (5, 6, 28, 29). Our
experiments, performed mostly in the absence of intracellular
K+and under conditions that would cause only mild intra-
cellular acidification, do not address whether this mechanism
contributes to sour transduction.
More recently, the TRP-channel related proteins PKD1L3/
PKD2L1 were identified in a subset of cells that mediate sour
taste, suggesting a role in sour transduction (3, 13, 14). Although
an initial report showed that the two subunits together formed
a Ca2+-permeable channel activated by severely acidic con-
ditions (pH <3), later experiments showed that activation of the
current occurred at a delay following washout of the stimulus
(17, 18). The contribution of PKD1L3 to sour is further called
into question by the observation that mice carrying a targeted
deletion of PKD1L3 have normal nerve and behavioral respon-
ses to sour stimuli (19). Our results from PKD1L3 KO animals
show that PKD1L3 does not directly contribute to the proton
current that we have described in PKD2L1-expressing cells (Fig.
2C). Because PKD2L1 requires assembly with PKD1L3 to traffic
normally (14, 40), these results also suggest that PKD2L1 does
not contribute to the proton conductance. This conclusion is
supported by our observation that the PKD1L3/PKD2L1 het-
eromer expressed in HEK cells did not respond to acid stimuli
used to evoke proton currents in taste cells (Fig. S3). It is still
possible that stronger acidification as used by Ishimaru et al. and
Inada et al. (14, 17), could regulate the gating of these channels,
An important question is whether the current carried by proton
entry is itself sufficient to drive the generation of action poten-
tials. We measured an inward current of 15.82 ± 2.03 pA at −80
mV in response to a drop in extracellular to pH 6, a stimulus that
was sufficient to evoke action potentials in cell-attached re-
cording. With a measured membrane resistance of 3–5 GΩ, this
would generate a membrane depolarization of >45mV, more
than enough to activate voltage-gated Na+channels (26). How-
ever, even though the charge carried by H+influx would be
sufficient to induce action potentials, we cannot exclude the pos-
sibility that, under physiological conditions, action potentials
are evoked synergistically by the current-mediated depolarization
and a depolarization that is a consequence of intracellular acid-
ification acting on leak potassium channels (28, 29).
In most sensory cells, transduction currents are carried by Na+
and Ca2+ions. Na+ions are relatively inert and therefore make
an ideal charge carrier, whereas Ca2+ions act specifically on
a host of Ca2+-binding proteins, tuning the sensory response.
Protons are rarely used as charge carriers for electrical signaling
in the nervous system, as high concentration of intracellular
protons are cell damaging (41). Why, then, would taste cells use
a proton channel to mediate sour transduction? Among sensory
neurons, the taste cell and the olfactory neuron face a unique
problem in that their apical surface is bathed in an extracellular
solution that undergoes extreme changes in ionic composition.
Olfactory responses of amphibians and fish persist in freshwater
because a significant component of the transduction current is
carried by outward movement of Cl−through Ca2+-activated Cl−
channels (42, 43). For sour taste transduction, a proton channel,
as opposed to a proton-gated Na+channel, would be able to track
changes in the concentrations of acids within the oral cavity,
without confounding contributions from Na+ions, which vary
widely in concentration and are detected separately as saltiness.
Materials and Methods
Generation of Transgenic Mice, Immunocytochemistry, and Single-Cell PCR.
Generation and validation of transgenic mice is described in SI Materials
Acute Dissociation of Taste Receptor Cells and Patch-Clamp Recording. Prep-
aration of mouse taste cells and patch clamp recording were previously
described (23).For whole-cell recording, the standard internal solution con-
tained the following (in mM): 120 CsAsp, 7 CsCl, 8 NaCl, 5 EGTA, 2.4 CaCl2
(100 nM free Ca2+), 2 MgATP, 0.3 Tris-GTP, and 10 Hepes, pH7.4; the stan-
dard bath solution was Tyrode’s solution. More information can be found in
SI Materials and Methods.
Flash Photolysis, Ca2+, and pH Imaging. Flash photolysis and Ca2+imaging were
performed as previously described (23). More information is provided in SI
Materials and Methods.
protons in PKD2L1-YFP cells. (A) Simultaneous measurement of the change
in intracellular Ca2+(Upper Left) and the magnitude of current (Lower Left)
in response to apical uncaging of NPE-caged proton in a PKD2L1-YFP cell.
Fluorescent images taken at different time points are shown at Right.
(B) Scatter plot of the change in intracellular Ca2+as a function of the in-
tegrated current in response to UV uncaging from experiments as in A. Red,
cells that fired action potentials; blue, cells that did not fire action poten-
tials; black, control data measured 10 s before the UV flash. (C) Proposed
model for sour taste transduction in PKD2L1-expressing cells. Pro-
ton entry through a proton-selective conductance specifically expressed
on apical surface of PKD2L1-expressing cells leads to depolarization and
generation of action potentials that propagate to the cell body and acti-
vate voltage-gated Ca2+channels. In addition to accessing this pathway,
weak acids may produce intracellular acidification and inhibit resting K+
Elevation of intracellular Ca2+in response to apical delivery of
| www.pnas.org/cgi/doi/10.1073/pnas.1013664107 Chang et al.
Suction Electrode Recording. To measure receptor currents, the apical surface
of a dissociated taste cell was drawn into a fire-polished pipette with a re-
sistance of 0.5–1 MΩ (∼3-μm diameter). The pipette contained 2 mM NPE-
caged proton (Tocris) in either Na+solution (in mM: 145 NaCl, 5 KCl, 1 MgCl2,
2 CaCl2, 20 dextrose, and 0.1 Hepes, pH 7.4) or Na+-free solution (in mM: 165
NMDG-Cl, 0.04 CaCl2, and 0.1 Hepes, pH 7.4). Other solutions are described in
SI Materials and Methods. The apical region was identified as a tapered
process that extended from the cell body (Fig. 1C). Cells used in this study
had similar or more elongated processes than the cell shown Fig. 1C. The seal
resistance was typically ∼5–10 MΩ. Liquid junction potentials were zeroed in
the bath before contact with the cell. Records were sampled at 5 kHz and
filtered at 1 kHz. When measuring the frequency of action potentials
evoked by the UV stimulus, only the cells that fired spontaneous APs were
included in the analysis.
ACKNOWLEDGMENTS. We thank N. Segil for technical help in generating
transgenic mice, H. Matsunami (Duke University Medical Center, Durham,
NC) for anti-PKD2L1 antiserum, D. Liu for help in performing single-cell PCR,
and D. Arnold and Y. Wang for careful reading of the manuscript. This work
was supported by National Institutes of Health Grant DC004564 (to E.R.L.).
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| vol. 107
| no. 51